NO317058B1 - Rapamycin derivatives, process for their preparation, pharmaceutical composition comprising such a derivative, kit comprising a preparation and use of the derivatives - Google Patents

Description

The present invention relates to rapamycin derivatives, a method for their preparation, their use as a pharmaceutical agent and pharmaceutical preparations containing these, as well as a kit or a package comprising a pharmaceutical preparation.

Rapamycin is a known macrolide antibiotic produced by Streptomvces h<yg>roscopicus which has the structure shown in formula A:

See e.g. McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) H3: 7433; US Patent No. 3,929,992. (Different numbering schemes have been proposed for rapamycin. To avoid confusion, when specific rapamycin derivatives are named herein, the names given are with reference to rapamycin using the numbering scheme of formula A.) Rapamycin is a potent immunosuppressant and is also has been shown to have antitumor and antifungal activity. However, its utility as a pharmaceutical agent is limited by its very low and variable bioavailability. Furthermore, rapamycin is insoluble and lacks stability, which makes it difficult to formulate stable galenic preparations.

Numerous derivatives of rapamycin are known. Certain 40-O-substituted rapamycins are described e.g. in US 5,258,389 and WO 94/09010 (O-alkyl rapamycins); WO 92/05179 (carboxylic acid esters), US 5,118,677 (amide esters), US 5,118,678 (carbamates), US 5

100,883 (fluorinated esters), US 5,151,412 (acetals) and US 5,120,842 (silyl ethers).

It has now surprisingly been discovered that certain new derivatives of rapamycin have an improved pharmacological profile compared to rapamycin and show greater stability.

According to the invention, a compound of formula I is provided

in which

R, is C 1-10 alkyl, C 3-10 alkynyl, hydroxy-C 3-10 alkynyl,

R 2 is a residue of formula II;

in which

R 3 is selected from H, C 1-4 alkyl, hydroxy-C 2-6 alkyl, hydroxy-C 1-6 alkoxy-C 2-6 alkyl

or C 1-6 -Alkoxy-C 2-6 -Alkyl;

R 4 is H or methyl;

Y is O,

X is OH or H,

R 1 is different from C 1-1 o-alkyl when X is OH.

Any "alk" unit or "alkyl" mentioned above may be branched, linear or cyclic; preferably it is a C1.6 aliphatic substituent optionally interrupted by an oxy linker, more preferably uninterrupted by oxy.

Preferred compounds are compounds of formula Ia

wherein R 1 is C 3 -alk-2-ynyl; R 2 is a residue of formula II as defined above and Y = O;

and of formula Ib

wherein R 1 is C 3 -alk-2-ynyl; R 2 is a residue of formula II as defined above and Y = O.

Particularly preferred compounds include

(i) 32-deoxo-rapamycin; (ii) 16-O-pent-2-ynyl-32-deoxo-rapamycin; (iii) 16-O-pent-2-ynyl-32(S)-dihydro-rapamycin; (iv) 16-O-pent-2-ynyl-32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin. Compounds of formula I may exhibit isomerism, and consequently additional isomeric forms will exist. It will be obvious that the present invention encompasses compounds of formula I, the individual isomers of formula F

where R 1 , R 2 , Y and X are as defined above, as well as isomeric mixtures thereof.

The individual isomers can be separated analogously in methods known in the art.

The present invention also provides a method for the preparation of the compounds of formula I which includes

a) to prepare a compound of formula I where X is H, reductive elimination of carbonyl at position 32 of a compound of formula IVa

where R 1 , R 2 and Y are as defined above, in protected or unprotected form,

and, if necessary, removing the protecting groups present; or

b) to prepare a compound of formula I wherein X is OH, stereoselective reduction of the carbonyl at position 32 of a compound of formula IVa as defined above; or c) converting a compound of formula I wherein R 1 is alkyl to provide a compound of formula I wherein R 1 is different from alkyl.

In method step a), the compound of formula IVa is preferably in protected form, i.e. it can include protective groups on functional groups that do not participate in the reactions, e.g. OH in position 28 and possibly in position 40 when R2 is a residue of formula n.

The reduction a) to the 32-deoxo compound of formula I can conveniently be carried out in two steps: i) by reacting a compound of formula IVa, preferably in protected form, with a hydride, e.g. diisobutylaluminum hydride or preferably lithium tri-tert-butoxyaluminum hydride, to prepare a corresponding 32-dihydro compound. Other methods and reagents known in the art for the reduction of ketones can be used to prepare the 32-dihydro compound from the corresponding ketone. They include e.g. hydrogenation, reduction with metals, metal hydride reduction, as described in "Comprehensive Organic Transformations," R.C. Larock, VCH Publishers Inc., New York, 1989, pp. 527-535, sections 7.1.1-7.1.4 and asymmetric reduction methods for ketones, e.g. as described in "Comprehensive Organic Transformations," R.C. Larock, VCH Publishers Inc., New York, 1989, pp. 540-547, section 7.1.15. The reduction step i) is then followed by

ii) conversion of the 32-dihydro compound into the corresponding 32-halogen derivative,

e.g. The 32-bromo or (preferably) 32-iodo derivative, which is then reduced e.g. by means of a hydride to the desired 32-deoxo derivative, and when required, deprotection of the resulting compound. Additional reagents used for the reduction of halides can be used and include e.g. metals

of low valence (ie, lithium, sodium, magnesium, and zinc) and metal hydrides (aluminum hydrides, boron hydrides, silanes, copper hydrides) (see "Comprehensive Organic Transformation," R.C. Larock, VCH Publishers, Inc., New York, 1989, pp. 18 -20, sections 1.5.1. and 1.5.2.).

Alternatively, halide reduction can be achieved using hydrogen or a source of hydrogen (ie formic acid or a salt thereof) in the presence of a suitable metal catalyst (Raney nickel, palladium metal or palladium complexes, complexes of rhodium or ruthenium) (see "Comprehensive Organic Transformations," R.C.Larock , VCH Publishers Inc., New York, 1989, pp. 20-24, section 1.5.3). Further known methods used for transforming an alcohol into the corresponding deoxy compound can also be used. These methods include e.g. direct reduction or reduction of an intermediate phosphorus compound, sulfonate, thiocarbonate, thiocarbamate or xanthate and is described e.g. in "Comprehensive Organic Transformations," R.C. Larock, VCH Publishers Inc., New York, 1989, pp. 27-31, sections 1.9.1.-1.9.4.

Suitable hydroxy protecting groups and methods for their formation and removal are known in the art, see e.g. "Protective Groups in Organic Synthesis", Second Edition T.W. Greene and P.GM. Wuts, John Wiley & Sons, New York, 1991, Chapter 2 and the references therein. Preferred OH-protecting groups are triorganosilyl groups, such as tri(C1.6)alkylsilyl (eg trimethylsilyl, triethylsilyl), triisopropylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, triarylsilyl (eg triphenylsilyl) or trialkylsilyl ( eg tribenzylsilyl). Deprotection can be carried out under mildly acidic conditions.

The reduction step i) can suitably be effected at a low temperature, e.g. from -10 to -80°C.

In step ii), the 32-dihydro compound is optionally converted in protected form, preferably the 32(R)-diastereoisomer, into an ester, preferably a sulphonate, e.g. mesylate, tosylate, nosylate or triflate, followed by displacement with a suitable halide, e.g. sodium iodide or bromide, tetrabutylammonium iodide or bromide, preferably in the presence of a base, e.g. an amine. The 32(R)-diastereoisomer can be separated from the mixture according to known separation techniques, e.g. chromatography.

Suitable hydrides for reducing the 32-halogen compound include e.g. radical hydrides, such as tributyltin hydride or tris-(trimethylsilyl)-silane. Reduction can also be carried out in the absence or presence of a radical initiator, e.g. 2,2'-azobisisobutyronitrile or preferably Et3B, conveniently at a temperature from 0 to 80°C.

An oxidizing agent, such as copper (H) acetate, can conveniently be added after the reduction step of i) or ii), if required, to selectively reoxidize to carbonyl an unwanted side reaction that may occur e.g. in position 9.

Alternatively, the 32-dihydro derivative can be directly converted to a halide by methods known in the art, e.g. by using triphenylphosphine in combination with N-bromo- or N-iodo-succinimide, carbon tetrabromide or tetraiodide, 1,2-dibromotetrachloroethane, 2,4,5-tribromo- or -triiodimidazole, iodine, 1,2-diiodoethane, or by use of thionyl bromide or methyltriphenoxyphosphonium iodide.

The reduction of the carbonyl at position 32 to the 32-deoxo derivative can also be carried out by the formation of a tosylhydrazone followed by treatment with a borane, e.g. catecholborane, or by the formation of a dithiane followed by suitable reduction, e.g. with Raney nickel or hydride, e.g. tributyltin hydride. Other known methods for transforming a ketone into the corresponding alkane can be used; such methods include e.g. direct reduction (see "Comprehensive Organic Transformations", R.C.Larock. VCH Publishers, Inc., New York, 1989, pp. 35-37, section 1.12.1.) or reduction via hydrazones ("Comprehensive Organic Transformations", R.C. Larock, VCH Publishers Inc., New York, 1989, pp. 37-38, section 1.12.2.) and via sulfur and selenium derivatives ("Comprehensive Organic Transformations", R.C. Larock, VCH Publishers Inc., New York, 1989, pp. 34-35, sections 1.10 and 1.11).

The reduction step b) of the 32(S)-dihydro compound of formula I is carried out under selected conditions. Preferably, a reducing agent is used which significantly promotes the reduction to 32(S), e.g. sodium triethylborohydride. The reduction can be appropriate

is carried out at a low temperature, e.g. from -50 to -80°C, in an inert solvent, e.g. THF diethyl ether, glyme, diglyme or methyl t-butyl ether. The separation of the 32(S)-dihydro compound from the low amounts of the 32(R)-dihydro compound produced can be carried out by methods known in the art, e.g. column chromatography, reverse phase chromatography.

If desired, the hydroxy groups in position 28 and possibly in position 40 can be protected before the reduction and then deprotected, e.g. as described above. Preferably, the reduction step b) is carried out without OH protection.

The transformation step c) can be carried out according to methods known in the art. For example, a compound of formula I, where Rj is alkyl, preferably methyl, can be reacted with a compound Rx-OH where Rx is alkenyl or hydroxyalkynyl to provide a compound of formula I where R, is alkynyl or hydroxyalkynyl. The reaction can conveniently be carried out in an aprotic solvent, e.g. dichloromethane, toluene, acetonitrile or THF under acidic conditions.

Preferably, the reduction is carried out in position 32, especially the reduction step b) on a compound of formula IVa where R1 already has the desired meaning, where e.g. Rx is alkynyl, thereby avoiding a later transformation after reduction. A compound of formula IVa where Rj is alkynyl or hydroxyalkynyl, used as starting material, can be prepared by using a compound Rx-OH as described above.

Compounds used as starting material can be prepared analogously to methods known and practiced in the art, e.g. as described in USP 5,258,389, WO 94/09010, WO 95/16691, USP 5,120,842, etc.

The following examples illustrate the invention. All temperatures are given in °C. The following abbreviations are used:

THF = tetrahydrofuran

TES = triethylsilyl

EXAMPLE 1;

32-Deoxorapamycin (R) = CH3; R2 = where R3 = H and R4 = CH3;

X = H; Y = 0)

To a stirred, cooled (-78°) solution of 26.1 g (22.85 mmol) of 28,40-bis-O-TES-rapamycin in 260 ml of THF is added 50.3 ml (50.3 mmol) of an IM solution of lithium-tri-t.-butoxyaluminum hydride in THF. The resulting mixture is allowed to warm to -15°C over two hours. The cooling bath is then replaced by an ice bath, which brings the temperature to 0°, and stirring is continued for 1 hour at this temperature. The reaction mixture is poured into a separatory funnel containing 750 ml of ethyl acetate and 400 ml of ice-cold 2N aqueous citric acid and shaken briefly. The aqueous layer is separated and extracted twice with cold ethyl acetate. The combined organic solution is washed with ice-cold 2N aqueous citric acid, water, saturated aqueous sodium bicarbonate and twice with saturated brine, then dried over anhydrous sodium carbonate, filtered and concentrated under reduced pressure. The residue, consisting of a mixture of 32(R)-dihydro-28,40bis-O-TES-rapamycin and (32R)9,32-bis-dihydro-28,40-bis-O-TES-rapamycin, dissolves without further purification in 260 ml of methanol. The solution is cooled to 0°C and treated with 6.85 g (34.31 mmol) of cupric (II) acetate. After stirring for one hour, the resulting suspension is diluted with methyl t-butyl ether and washed twice with water and twice with saturated saline solution. The aqueous layers are back-extracted with methyl t-butyl ether. The combined organic solution is dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by silica gel chromatography (60:40 hexane/methyl-t-butyl ether) to give pure 32(R)-dihydro-28,40-bis-O-TES-rapamycin as a white solid.

<]>H NMR (CDC13) 4:1 mixture of rotamers, chemical shifts in parentheses refer to the smallest rotamer 6 0.72(1H, dd, H-38ax), 1.63 (1.60) (3H, s , C17-CH3), 1.66 (1.69) (3H, s, C29-CH3), 1.77 and 1.81 (H-33), 2.46 (1H, m, H-31), 2.82 (2.91) (1H, m, H-25), 2.91 (1H, m, Y-39), 3.13 (3, s, Cl6-OCH3), 3.26 (3H, s, C27-OCH3), 3.41 (1H, m, H-40), 3.43 (3H, s, C39-OCH3), 3.62 (1H, m, H-32), 3.75 ( 3.57) (1H, d, H-27), 4.10 (1H, d, H-28), 4.81( 1H, broad s, C10-OH), 5.05 (1H, d, H -34), 5.27 (1H, d, H-30), 5.36 (1H, d, H-2), 5.69 (1H, dd, H-22), 6.03 (5.96 ) (1H, d, H-18), 6.15 (1H, dd, H-21), 6.33 (1H, dd, H-20), 6.40 (1H, dd, H-19)

MS (FAB, Lii matrix) m/z 1150 ([M + Lif) (rel. intensity 100)

To a stirred, cooled (-15°C) solution of 20.69 g (18.10 mmol) of 32(R> dihydro.28,40-bis-O-TES-rapamycin and 7.55 ml (54.27 mmol) of triethylamine in 200 mL of methylene chloride, 2.10 mL (27.02 mmol) of methanesulfonyl chloride is added. The mixture is stirred for 20 minutes, then diluted with ethyl acetate and saturated aqueous sodium bicarbonate is added. The layers are separated and the aqueous layer is extracted three times with ethyl acetate. The combined organic phase is washed with saturated aqueous sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue can be purified by column chromatography on silica gel (80:2 hexane/ethyl acetate) to give pure 32(R) -dihydro-32-0-mesyl-28,40-bis-O-TES-rapamycin as a white solid, but routinely the crude product is used in the subsequent step without further purification.

<*>H NMR (CDCl3) δ 0.77 (1H, dd, H-38ax), 1.67 (3H, s, C17-C3), 1.72 (3H, s, C29-CH3), 2, 77 (1H, M, H-25), 2.92 (1H, m, H-39), 3.03 (3H, s, C16-OCH3), 3.17 (3H, s, C27-OCH3), 3.21 (3H, s, C39-OCH3), 3.42 (1H, m, H-40), 3.45 (3H, s, CH3SO3), 3.91 (1H, d, H-27), 4.10 (1H, d, H-28), 4.72 (1H, m, H-32), 4.94 (1H, s, C10-OH), 5.12 (1H, m, H-34 ), 5.25 (1H, d, H-30), 5.43 (1H, d, H-2), 5.88 (1H, dd, H-22), 6.03 (1H, d, H -18), 6.18 (1H, dd, H-21), 6.37 (1H, dd, H-20), 6.44 (1H, dd, H-19)

MS (FAB, Lii matrix) m/z 1228 ([M + Li]<+>) (rel. intensity 68), 1132 ([M - CH3S=3H) + Li]<4>) (rel. intensity 100)

A mixture of 22.35 g (18.30 mmol) of 32(R)-dihydro-32-O-mesyl-28,40-bis-O-TES-rapamycin, 27.50 g (183.33 mmol) of sodium iodide and 6.3 mL (36.68 mmol) in isopropylethylamine in 400 mL THF is heated to reflux for 6 h, then allowed to cool to room temperature. The resulting mixture is diluted with ethyl acetate and treated with 38.4% aqueous sodium bisulfite. The teams are separated. The organic phase is washed three times with saturated aqueous sodium bicarbonate and once with saturated saline solution, then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (83:17 hexane/ethyl acetate) to give pure 32(S)-deoxo-32-iodo-28,40-bis-O-TES-rapamycin.

<*>H NMR (CDC13) 1.5:1 mixture of rotamers, chemical shifts in parentheses refer to the minor rotamer 8 0.73 (1H, dd, H-38ac), 1.68 (1.66) 6H, s, C17-CH3 and C29-CH3), 2.72 (1H, m, H25), 2.91 (2H, m, H-32 and H-39) 3.15 (3H, s, C16-OCH3) 3.30 (3.31) (3H, s, C27-OCH3), 3.43 (3.41) (3H, s, C30-OCH3), 3.77 (3.91) 1H, d, H- 27), 4.21 (4.25) (1H, d, H-28), 4.51 (1H, s, C10-OH), 5.45 (5.48) 11H, d, H-30) , 5.60 (5.79) (1H, dd, H-22), 6.02 (5.85) 1H, d, H18)

MS(FAB, Lii matrix) m/z 1260 ([M + Li]"<1>) (rel. Intensity 100)

To a stirred, cooled (0°) solution of 16.79 g (13.9 mmol) of 32(S)-deoxo-32-iodo-28,40-bis-O-TES-rapamycin in 190 ml of toluene is added 7 mL (26.38 mmol) of tributyltin hydride followed by 1.3 mL (1.30 mmol) of a 1M solution of triethylborane in hexane. The mixture is stirred for 30 minutes and quenched with saturated aqueous ammonium chloride. The layers are separated and the aqueous layer is extracted twice with ethyl acetate. The combined organic layers are washed with water, saturated aqueous sodium bicarbonate, water and three times with saturated brine, then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (75:22 hexane/methyl-t-butyl ether) to give pure 32-deoxo-28,40-bis-O-TES-rapamycin as a white solid.

'h NMR (CDCl 3 ) 2.5:1 mixture of rotamers, chemical shifts in parentheses refer to the minor rotamer 8 0.73 (1H, dd, H-38ax), 1.62 (1.57) (3H, s , C17-CH3), 1.68 (1.72) 3H, s, C29-CH3), 2.77 (2.91) (1H, m, H-25), 2.91 (1H, m, H -39), .15 (3H, s, C16-OCH3), 3.70 (3.67) (1H, d, H-27), 4.11 (4.07) (1H, d, H-28 ), 4.57 (1H, broad s, C10-OH), 4.87 (4.67) 1H, d, H-34), 5.19 (5.08) (1H, d, H-30) , 5.32 (1H, d, H-2), 5.60 (5.66)

(1H, dd, H-22), 6.01 (5.92) (1H, d, H-18), 6.17 (1H, dd, H-21), 6.30 (1H, dd, H -20), 6.40 (1H, dd, H-19)

MS (FAB, Lii matrix m/z 1134 ([M + Li]<*>) (rel. intensity 100)

To a stirred, cooled (-15°C) solution of 10.73 g (9.52 mmol) of 32-deoxo-28,40-bis-O-TES-rapamycin in 85 ml of methanol is added dropwise 9.5 ml 2N aqueous sulfuric acid. After the addition is complete, the reaction mixture is warmed to 0° and stirred for 1.5 hours, then diluted with ethyl acetate and "quenched" with saturated sodium bicarbonate. The layers are separated and the aqueous layer is extracted with three portions of ethyl acetate. The combined organic phase is washed three times with saturated sodium bicarbonate and with brine, then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is dissolved in diethyl ether, whereupon the desired 32-deoxorapamycin is crystallized (colorless crystals).

<*>H NMR (CDC13) 3:1 mixture of rotamers, chemical shifts in parentheses refer to the minor rotamer 8 0.70 (1H, dd, H-38ac), 1.14 and 1.32 (H32), 1 .56 (H-33), 1.65 (1.62) (3H, s, C17-CH3), 1.68 (1.70) (3H, s, C29-CH3), 2.31 (2H, m, H-23 and H-31), 2.82 (2.95) (1H, m, H-25), 2.95 (1H, m, H-39), 3.14 (3H, s, C16-OCH3), 3.32 (3H, s, C27-OCH3), 3.38 (1H, m, H-40), 3.43 (3.41) (3H, s, C39-OCH3), 3 .61 (1H, d, H-27), 4.12 (1H, d, H-28), 4.80 (4.71) (1H, d, H-34), 5.22 (1H, d , H-30), 5.21 (1H, d, H-2), 5.56 (1H, dd, H-22), 5.95 (5.87) (1H, d, H-18), 6.16 (1H, dd, H-21), 6.36 (1H, dd, H-20), 6.41 (1H, dd, H-19)

MS (FAB, Lii matrix) m/z 906 ([M + Li]<*>) (rel. intensity 100)

Example 2: 16-pent-2-yloxy-32(S)-dihydro rapamycin (Rj = pent-2-ynyl; R2 = n where R3 = H and R4 = CH3; X = OH; Y = 0)

To a stirred, cooled (0°) solution of 970 mg (1.06 mmol) of 32(S)-dihydro-rapamycin and 1.39 mL (15.00 mmol) of 2-pentyn-1-ol in 20 mL methylene chloride, 0.50 ml (6.50 mmol) of trifluoroacetic acid is added. The mixture is stirred at 0° for three hours and "quenched" with saturated aqueous sodium bicarbonate. The layers are separated, and the aqueous layer is extracted with three portions of ethyl acetate. The combined organic solution is washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude mixture is purified by column chromatography on silica gel (20:80 hexane-ethyl acetate), then by reverse-phase HPLC (RP18, 81:19 methanol-water) to give the title compound as a white amorphous solid.

'H NMR (CDCl 3 ) 2.5:1 mixture of rotamers, chemical shifts in parentheses refer to the minor rotamer 8 0.71 (1H, dd, H-38 ac), 1.14 (1.05) (3H, t, CH3CH2CCCH20), 1.67 (3H, s, 17-CH3), 1.69 (3H, s, 29-CH3) 2.21 (sH, qt, CH3CH2CCCH20), 2.96 (1H, m, H -39), 3.33 (3.37) (3H, s, 27-OCH3), 3.41 (3.39) (3H, S, 39-OCH3), 3.78 (1H, dt, CH3CH2CCCH/ /0), 4.0 (1H, dt, CH3CH2CCHHO), 5.52 (5.71) (1H, dd, H-22), 5.98 (5.83) (1H, d, H-18) , 6.15 (1H, m, H-21), 6.30 (1H, dd, H-20), 6.40 (1H, dd, H-19) MS (FAB) m/z 974 ([M +Li]<*>)

Example 3: 16-pent-2-ynyloxy-32(S)-dihydrorapamycin (alternative method)

Rapamycin is reacted with 2-pentyn-1-ol in a method analogous to that in example 2 to give 16-pent-2-ynyloxy-rapamycin.

To a stirred, cooled (-77°) solution of 17.5 g (18.1 mmol) of 16-demethoxy-16-pent-2-ynyloxy-rapamycin in 180 ml of THF is added 21.7 ml (21.7 mmol) of a 1M solution of sodium triethylborohydride in THF. After 1 hour at -77°, the reaction is stopped and neutralized with an aqueous solution of 10% citric acid. The reaction mixture is then allowed to come to room temperature, and most of the THF is removed by evaporation under reduced pressure. The resulting solution is extracted twice with ethyl acetate, the organic phases are combined and dried over sodium sulfate. After evaporation of the solvent, the crude reaction product is chromatographed over silica gel by elution with hexane/acetone 7/3. Final purification is achieved by preparative HPLC (RP-18, 76:24 methanol:water) to give the title compound as a white amortized solid.

The spectral data are identical to those obtained by the second method.

Example 4: 32(SVdihydro-40-O-(2-methoxysv)eM-rapamcin (R 1 = CH 3 ; R 2 =n

where R3 = 2-methoxyethyl) and R4 = CH3; X = OH; X = O)

To a stirred, cooled (0°) solution of 2.17 g (2.00 mmol) of 40-O-(2-methoxy)ethyl-28-O-TES-rapamycin in 20 ml of THF is added dropwise 4.4 ml (4.4 mmol) of an IM solution of "L-selectride" in THF. The resulting yellow solution is stirred for three hours at 0° and the excess hydride reagent is quenched by the addition of 2 ml of MeOH. The solution is diluted with methyl t-butyl ether and saturated aqueous Rochelle's salt solution is added. This mixture is allowed to warm to room temperature and stirring is continued for 15 minutes. The layers are separated and the organic solution is washed with cold 1N HCl, saturated saline, 1N sodium bicarbonate and again with saline. The aqueous washing solutions are back-extracted with methyl-t-butyl ether. The combined organic layers are dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude mixture of the 32S and 32R isomers of 32-dihydro.40-O-(2-methoxy)ethyl-28.O-TES-rapamycin.

The crude product obtained above is dissolved in 20 ml of acetonitrile and cooled to 0°. To the resulting solution is added 2 ml of HF-pyridine complex. Stirring is continued for 1 hour and IN sodium bicarbonate is added. The mixture is extracted three times with methyl t-butyl ether. The combined organic solution is washed with IN sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Purification is carried out by reverse phase HPLC (RP 18.5 5(j,m, 50:50-100:0 acetonitrile-water over 60 minutes) to give 32(S)-dihydro-40-O-(2-methoxyethyl- rapamycin and 32(R)-dihydro-40-O-(2-methoxy)ethyl-rapamycin as by-product.

32(S)-dihydro-40-O-(2-methoxy-ethyl-rapamycin: <*>H NMR (CDC13) 2:1 mixture of rotamers, the chemical shift in parentheses refers to the smaller rotamer 5 0.77 (1H , dd, H-38 ax), 1.67 (H, s, C17-CH3 and C29-CH3), 2.50 (1H, m, H-31), 3.01 (1H, m, H-25 ), 3.12 (2H, m, H-39 and H-40), 3.14 (3.15) (3H, s, OCH3), 3.28 (1H, m, H-32), 3, 36 (3.34) (3H, 2, OCH3), 3.39 (3.38) (2H, s, OCH3), 3.48 (3.46) (3H, s, OCH3), 3.55 and 3.75 (4H, 2m, OCH2CH2O), 3.84 (1H, m, H-14), 4.12 (4.16) (1H, d, H-28), 4.73 (1H, s, C10-OH), 5.03 (1H, m, H-34)

MS (FAB) m/z 980 ([M + Li]<*>)

Example 5: (32S)-dihydro-40-O-(2-hydroxy)ethyl-rapamycin (R 1 = CH 3 ; R 2 =H

where R 3 = CH 2 CH 2 OH and R 4 = CH 3 ; X = OH; Y = O)

By following the procedure in example 4, but by using the suitable starting material, the compound in the title is obtained.

(32S)-dihydro-40-O-(2-hydroxy)ethyl-rapamycin: <]>H NMR (CDC13) 1.7:1 mixture of rotamers, chemical shifts in parentheses refer to the smaller rotamer 8 0.76 ( 1H, dd, H-38ax), 2.50 (1H, m, H-31), 3.10 (1H, m, H-39), 3.13 (3.14) (3H, s, C16- OCH3), 3.20 (1H, m, H.40), 3.28 (1H, m, H-32), 3.36 (3.38) (3H, s, C27-OCH3), 3.45 (3.43) (3.41) (3H, s, C39-OCH3), 3.50 (1H, d, H-27), 3.58 and 3.70 (4H, m, OCH2CH2OH), 4, 12 (4.16) (1H, d, H-28), 5.06 (1H, m, H-34), 5.60 (1H, dd, H-22), 5.99 (1H, d, H-18), 6.17 (1H, dd, H-21), 6.33 (1H, dd, H-20), 6.42 (1H, dd, H-19)

MS (FAB, Lii matrix) m/z 966 ([M + Li]<*>) (rel. intensity 100)

Example 6: 16-pent-2-ynyloxy-32-deoxo rapamycin (R) = pent-2-ynyl; R2 = II

where R 3 = H and R 4 = CH 3 ; X = H;

Y = 0)

By following the procedure in Examples 1 and 2 or 3, but using the appropriate starting materials, the title compound is obtained.

1H NMR (CDCl 3 ) δ 0.70 (1H, dd, H-38ax), 1.23 (3H, t, CH3CH2CCCH2O), 2.21 (2H, ddq, CH3CH2CCCH2O), 2.78 (1H, m, H-25), 2.94 (1H, m, H-39), 3.31 (3H, s, C27-OCH3), 3.41 (3H, s, C39-OCH3), 3.62 (1H, d, H-27), 3.78 (1H, ddd, CH3CH2CCCH20), 4.02 (1H, ddd, CH3CH2CCCH20), 4.12 (1H, d, H-28), 4.79 (1H, m, H-4), 5.20 (1H, d, H.30), 5.28 (1H, wide d, H-2), 5.50 (lh, dd, H-22), 5.97 (1H , d, H-18), 6.14 (1H, dd, H-21), 6.30 (1H, dd, H-20), 6.38 (1H, dd, H-19)

MS (FAB, Lii matrix m/z ([M + Li]<*>) (rel. intensity 100)

The compounds of formula I exhibit pharmaceutical activity and are therefore useful as pharmaceutical agents.

In particular, the compound of formula I has immunosuppressive and antiproliferative activity as indicated in the following in vitro and in vivo test methods.

1. Mixed lymphocyte reaction (MLR)

The mixed lymphocyte reaction was originally developed in connection with allografts, to assess tissue compatibility between potential organ donors and recipients, and is one of the best established models of immune reaction in vitro. A murine model MLR, e.g. as described by T. Meo in "Immunological Methods", L. Lefkovits and B. Pemis, editors, Academic Press, N.Y. pages 227-239 (1979), is used to demonstrate the immunosuppressive effect of the compounds of formula I. Spleen cells (2 x 10 wells) from Balb/c mice (female mice, 8 - 10 weeks) are coincubated in microtiter plates for 5 days with 0.5 x IO6 irradiated (2000 rad) or mitomycin C-treated spleen cells from CBA mice (female mice, 8-10 weeks). The irradiated allogeneic cells induce a proliferative response in the Balb/c spleen cells which can be measured by means of labeled precursor incorporation into DNA. Since the stimulator cells are irradiated (or mitomycin C-treated), they do not respond to Balb/c cells with proliferation, but they retain their antigenicity. The antiproliferative effect of the compounds of formula I on the Balb/c cells is measured at different dilutions and the concentration resulting in 50% inhibition of cell proliferation (IC50) is calculated. The inhibitory capacity of the test sample can be compared to rapamycin and expressed as a relative IC50 (ie IC50 test sample/IC50 rapamycin). The compounds in Examples 1 and 2 of this test are found to have a relative IC 0 of 0.3 and 0.08, respectively.

2. IL-6 mediated proliferation (IL-6PROL)

The abilities of the compounds of formula I to interfere with growth factor associated signaling pathways are assessed using an interleukin-6 (IL-6) dependent mouse hybridoma cell line. The analysis is carried out in 96-well microtiter plates. 5000 cells/well are cultured in serum-free medium (as described by M. H. Schreier and R. Tees in Immunological Methods, I, Lefkovits and B. Pernis, Eds., Academic Press 1981, Volume II, pages 263-275), supplemented with 1 ng recombinant IL-6/ml. After 66 hours of incubation in the absence or presence of a test sample, the cell is pulsed with 1 1 Ci (3-H)-thymidine/well for a further 6 hours, harvested and counted by liquid scintillation. (3-H)-thymidine incorporation into DNA correlates with the increase in cell number and is therefore a measure of cell proliferation. A dilution series of the test sample allows calculation of the concentration resulting in 50% inhibition of cell proliferation (IC50). The inhibitory capacity of the test sample can be compared to rapamycin and expressed as a relative IC50 (ie, test sample/IC 50 rapamycin). The compounds in examples 1 and 2 are found in this test to have a relative IC50 of 0.2 and 0.09 respectively.

3. Macrophilin binding assay (MBA)

Rapamycin and the structurally related immunosuppressive agent, FK-506, are both known to bind in vivo to macrofilin-12 (also known as FK-506 binding protein or FKBP-12), and this binding is thought to be associated with the immunosuppressive activity of these compounds. The compounds of formula I also bind strongly to macrophilin-12, as demonstrated in a competitive binding assay. In this assay, FK-506 coupled to BSA is used to coat microtiter wells. Biotinylated recombinant human macrophilin-12 (biot-MAP) is allowed to bind in the presence or absence of a test sample to that in mobilized FK-506. After washing (to remove non-specifically bound macrophilin), bound biot-MAP is assessed by incubation with a streptavidin-alkaline phosphatase conjugate, followed by washing and subsequent addition of p-nitrophenyl phosphate as a substrate. The reading is OD at 405nm. Binding of a test sample to biot-MAP results in a decrease in the amount of biot-MAP bound to FK-506 and consequently in a decrease in OD405. A dilution series of the test sample allows determination of the concentration resulting in 50% inhibition of biot-MAP binding to the immobilized FK-506 (IC50). The inhibitory capacity of a test sample is compared to the IC50 of free FK506 as a standard and expressed as a relative IC50 (ie, IC50 test sample/IC50 free FK506). In this analysis, the compounds according to examples 1,2 and 5 are found to have a relative IC50 of 1,2,8 and 2.5 respectively.

4. Localized Graft-versus-Host (GvH) reaction

In vivo efficacy of the compounds of formula I is demonstrated in a suitable animal model, e.g. as described in Ford et al, TRANSPLANTATION K) (1970) 258. Spleen cells (1 x IO<7>) from 6-week-old female Wistar/Furth (WF) rats are injected subcutaneously on day 0 into the left hind paw of (F344 x WF) Fj female rats weighing approx. 100 g. The animals are treated for 4 consecutive days and the popliteal lymph nodes are removed and weighed on day 7. The difference in weight between the two lymph nodes is taken as the parameter for evaluating the reaction.

5. Renal allograft reaction in the rat

A kidney from a DA(RTl<a>) or Brown-Norway (BN) (RTl<n>) donor rat is transplanted onto the renal vessel of a unilateral (left side) nephrectomized (Lewis RT1') recipient rat using an end- end-to-end anastomosis. The ureteral anastomosis is also end-to-end. Treatment starts on the day of transplantation and is continued for 14 days. A contralateral nephrectomy is performed 7 days after transplantation, leaving the recipient as dependent on the performance of the donor kidney. Survival of the graft recipient is considered a parameter for a functional graft.

6. Experimentally induced allergic encephalomyelitis (EAE) in rats

Efficacy of the compound of formula I in EAE is measured e.g. by the method described in Levine & Wenk, AMER J PATH 47 (1965) 61; McFarlin et al, JIMMUNOL H3 (1974) 712; Borel, TRANSPLANT. & CLIN. IMMUNOLH

(1981) 3. EAE is a widely accepted model of multiple sclerosis. Male Wistar rats

injected into the hind paw with a mixture of bovine spinal cord and complete Freund's adjuvant. Symptoms of the disease (paralysis of the tail and both hind legs) usually develop within 16 days. The number of sick animals as well as the time of onset of the disease are recorded.

7. Freund's adjuvant arthritis

Efficacy against experimentally induced arthritis is shown by using the method described e.g. i Winter «fe Nuss, ARTHRITIS & RHEUMATISM 9

(1966) 394; BILLINGHAM & DAVIES, HANDBOOK OF EXPERLMENTAL

PHARMACOL (Vane & Ferreira Eds, Springer-Verlag, Berlin) 50/11 (1979) 108-144. OFA and Wistar rats (male or female, 150 g body weight) injected i.c. at the root of the tail or in the hind paw with 0.1 ml of mineral oil containing 0.6 mg of lyophilized heat-killed Mycobacterium smegmatis. In the developing arthritis model, treatment is started immediately after injection of the adjuvant (days 1 - 18); in the established arthritis model, treatment is started on day 14, when the secondary inflammation is well developed (days 14 -20). At the end of the experiment, the swelling of the joints is measured using a micro caliper. The ED50 is the oral dose in mg/kg that reduces the swelling (primary or secondary) to half that of the control animals.

8. Antitumor and MDR activity

The antitumor activity of the compounds of formula I and their ability to promote the action of antitumor agents by attenuating multidrug resistance is demonstrated e.g. when administering an anti-cancer agent, e.g. colchicine or etoposide to a multidrug-resistant cell and drug-sensitive cells in vitro or to animals having multidrug-resistant or drug-sensitive tumors or infections, with or without co-administration of the compounds of formula I to be investigated, and by administration of the compound of formula I alone.

Such in vitro testing is performed using any suitable drug-resistant cell line and control (parenteral) cell line, generated e.g. as described by Ling et al., J. Cell. Physiol. 83,103-116 (1974) and Bech-Hansen et al. J. Cell. Physiol. 88,23-32 (1976). Particular clones selected are the multi-drug resistant (eg colchicine resistant) line CHR (subclone CSS3.2) and the

parenteral, sensitive line AUX Bl (subclone AB1 Sil).

In vivo antitumor and anti-MDR activity is shown e.g. in mice injected with multidrug-resistant and drug-sensitive cancer cells. Ehrlich peritoneal carcinoma (EA) sublines resistant to drug DR, VC, AM, ET, TE or CC are developed by stepwise transfer of EA cells to subsequent generations of BALB/c host mice according to the methods described by Slater et al., J. Clin . Invest, 70, 1131 (1982).

Equivalent results can be obtained by using the compounds of formula 1 in experimental models of comparable performance, e.g. in vitro, or by using laboratory animals infected with drug-resistant or drug-sensitive viral strains, antibiotic (eg penicillin) resistant and sensitive bacterial strains, antifungal resistant and sensitive fungal strains as well as drug-resistant protozoal strains, e.g. plasmodial strains, e.g. naturally occurring substrains of Plasmodium falciparum exhibiting acquired chemotherapeutic, antimalarial drug resistance.

9. Mip and Mip-like factor inhibition

In addition, the compounds of formula I bind and block a variety of Mip (macrophage infectivity potentiator) and Mip-like factors, which are structurally similar to macrophilin. Mip and Mip-like factors are virulence factors produced by a wide variety of pathogens, including those of the genus Chlamydia. e.g. Chlamydiatrachomatis: Neisseria. e.g. Neisseria meningitidis: and Legionella, e.g. Legionella pneumophilia; and also by the obligate parasitic members of the order Rickettsiales. These factors play a critical role in the establishment of intracellular infection. The efficacy of the compound of formula I in reducing the infectivity of pathogens producing Mip or Mip-like factors can be demonstrated by comparing the infectivity of pathogens in cell culture in the presence and absence of the macrolides, e.g. using the methods described in Lundemose, et al., Mol. Microbiol. (1993) 7: 777.

10. Chronic allograft wasting

The kidney from a DA (RTl<a>) male rat is orthotopically transplanted into a Lewis (RT1<1>) male recipient. A total of 24 animals are transplanted. All animals are treated with cyclosporin A at 7.5 mg/kg/day per os for 14 days starting on the day of transplantation, to prevent acute cellular rejection. Contralateral nephrectomy is not performed. Each experimental group treated with a distinct dose of a compound of formula I or placebo comprises six animals.

At the start of day 53-64 after transplantation, the recipient animals are treated per os for an additional 69-72 days with a compound of formula I or receive placebo. At 14 days after transplantation, the animals are subjected to graft assessment by magnetic resonance imaging (MRI) with perfusion measurement of kidneys (with comparison of the grafted kidney and the own contralateral kidney). This is repeated at days 53-64 after transplantation and at the end of the experiment. The animals were then necropsied. The rejection parameter such as MRI scoring, relative perfusion rate of the grafted kidney and histological scoring of the renal allograft for cellular rejection and vascular changes are determined and analyzed statistically. Administration of a compound of formula I, e.g. the compound according to example 1 or 2, at a dose of 0.5 to 2.5 mg/kg in this rat kidney allograft model produces a reduction in all the above-mentioned de-icing parameters.

11. Angioplasty

Balloon catheterization is performed on day 0, essentially as described by Powell et al. (1989). Under "Isofluorane" anaesthesia, a "Fogarty 2F" catheter is introduced into the left common carotid artery via the external carotid and inflated (distension æ 10 jai H20). The unlocked balloon is retracted along the length of the common carotid three times, the last two times under gentle twisting to achieve a uniform de-endothelialization. The catheter is then removed, a ligature is placed around the external carotid to prevent bleeding and the animals are allowed to recover. 2 groups of 12 RoRo rats (400 g, approximately 24 weeks old) are used for the investigation: a control group and a group receiving the compound to be investigated. The rats are completely randomized during all handling, experimental procedures and analyses.

The connection to be examined is administered p.o. (gavage) at the start 3 days before balloon injury (day -3) until the end of the examination, 14 days after balloon injury (day +14). Rats are kept in individual cages and given access to food and water ad Iibidum.

The rats are then anesthetized with "Isofluorane", a perfusion catheter is inserted through the left ventricle and secured in the aortic arch, and an aspiration cannula is inserted into the left ventricle. Animals are perfused under a perfusion pressure of 150 mmHg, initially for 1 min. with 0.1 M phosphate-buffered saline solution (PBS, pH 7.4) and then for 15 min. with 2.5% glutaraldehyde in phosphate buffer (pH 7.4). The perfusion pressure is 105 mmHg at the tip of the cannula ("100 mmHg in the carotid artery, as determined in a preliminary experiment by inserting a cannula attached to a pressure transducer in the external carotid). Carotid arteries are then cut open, separated from surrounding tissue and immersed in 0.1 M cacodylate buffer (pH 7.4) containing 7% sucrose and incubated overnight at 4°C. The following day, the carotid is immersed and shaken for 1 hour at room temperature in 0.05% KMnO 4 in 0.1 M cacodylate. The tissues are then dehydrated in a graded ethanol series; 2x10 min in 75%, 2x10 min in 85%, 3x10 min in 95% and 3x10 min in 100% ethanol. The dehydrated carotids are then embedded in 'Teknovit 7100" according to the manufacturer's recommendations. The embedding medium is allowed to polymerize overnight in a desiccator under argon, since oxygen has been found to inhibit proper curing of the blocks.

Sections of thickness 1-2 µm are cut from the midsection of each carotid with a hard metal knife on a rotary microtome and stained for 2 min with Giemse stain. About. 5 sections from each carotid are repaired in this way and the cross-sectional area of the media, neointima and lumen is assessed morphometrically using an image analysis system (MCID, Toronto, Canada). In this assay, the compounds of formula I, e.g. the compound of Example 1 or 2, myointimal proliferation when administered orally at a daily dose of from 0.5 to 2.5 mg/kg.

The compounds of formula I are also useful in assays to detect the presence or amount of macrophilin compounds, e.g. in competitive assays for diagnostic or screening purposes. Accordingly, compounds of formula I can be used as screening tools to determine the presence of macrophilin-binding compounds in a test solution, e.g. blood, blood serum or test fluid to be screened. Preferably, a compound of formula I is immobilized in microtiter wells and then allowed to bind in the presence or absence of a test solution to labeled macrophilin-12 (FKBP-12). Alternatively, FKBP-12 is immobilized in microtiter wells and allowed to bind in the presence and absence of a test solution to a compound of formula I which is labeled, e.g. fluorine-, enzymatically- or radiolabelled, e.g. a compound of formula I wherein R[ includes a labeling group. The plates are washed and the amount of bound, labeled compound is measured. The amount of macrophilin-binding substance in the test solution is approximately inversely proportional to the amount of bound labeled compound. For quantitative analyses, a standard binding curve is generated using known concentrations of macrophilin binding compound.

The compounds of formula I are therefore useful in connection with the following conditions:

a) Treatment and prevention of acute or chronic organ or tissue transplant rejection, e.g. the processing of recipients of e.g. heart, lung, combined

heart-lung, liver, kidney, pancreas, skin or cornea transplants. They are also indicated for the prevention of graft-versus-host disease, such as that resulting from bone marrow transplantation.

b) Treatment and prevention of graft vasculopathies, e.g. atherosclerosis.

c) Treatment and prevention of smooth muscle cell proliferation and migration leading to tissue intimal thickening, blood vessel obstruction, obstructive coronary atherosclerosis, restenosis. d) Treatment and prevention of autoimmune disease and of inflammatory conditions, in particular inflammatory conditions with an etiology that includes a

autoimmune component, such as arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and rheumatic disease. Specific

autoimmune diseases for which the compounds of formula I can be used include autoimmune hematological conditions (including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma, Wegener granulomatosis, dermatomysis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic psilosis, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis and Crohn's disease), endocrinopathy, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I) uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, glomerulonephritis (with and without nephrotic syndrome, eg including idiopathic nephrotic syndrome or minimal change nephropathy) and juvenile dermatomyosis.

e) Treatment and prevention of asthma.

f) Treatment of multi-drug resistance (MDR). The compounds of formula I suppress P-glycoproteins (Pgp), which are the membrane transport molecules associated with MDR. MDR is particularly problematic in cancer patients and AJDS patients who do not respond to conventional chemotherapy because the drug is pumped out of the cells by Pgp. The compounds of formula I are therefore useful in promoting the efficacy of other chemotherapeutic agents in the treatment and control of multi-drug resistance conditions such as multi-drug-resistant cancer or multi-drug-resistant AIDS. g) Treatment of proliferative conditions, e.g. tumors, hyperproliferative skin diseases and the like.

h) Treatment of fungal infections.

i) Treatment and prevention of inflammation, especially when potentiating

the effect of steroids.

j) Treatment and prevention of infection, especially infection by pathogens that have Mip or Mip-like factors.

For the above-mentioned indications, the required dose will naturally vary, e.g. depending on the condition to be treated (e.g. the type of disease or the nature of

the resistance), the desired effect and the method of administration. In general, however, satisfactory results are obtained when administered orally at dosages of the order of 0.05 to 5 or up to 10 mg/kg/day, e.g. of the order of 0.1 to 2 or up to 7.5 mg/kg /day administered once or, in divided doses, 2 to 4 times per day, or when administered parenterally, e.g. intravenously, e.g. by i.v. drip or infusion, at doses of the order of 0.01 to 2.5 up to 5 mg/kg/day, e.g. of the order of magnitude from 0.05 to 0.1 up to 1.0 mg/kg/day. Suitable daily doses for patients are therefore of the order of 500 mg p.o., e.g. of the order of 5 to 100 mg p.o., or of the order of 0.5 to 125 up to 250 mg i.v., e.g. of the order of magnitude from 2.5 to 50 mg i.v..

Alternatively, and even preferably, the dosage is arranged in a patient-specific manner in order to provide pre-determined regular ongoing ductal blood levels as measured by RIA of the order of magnitude from 50 or 150 up to 500 or 1000 ng/ml, i.e. analogous to methods for dosing cyclosporine immunosuppressants used today treatment.

The compounds of formula I can be administered as the only active ingredient or together with other drugs. For example, in immunosuppressive applications such as a prevention and treatment of graft versus host disease, graft rejection or autoimmune disease, the compounds of formula I can be used in combination with cyclosporins or ascomycins, or their immunosuppressive analogues, e.g. cyclosporine A, cyclosporine G, FK-506, etc.: corticosteroids; cyclophosphamides; azathioprine; methotrexate; brequinar, leflunomide; mizoribine, mycophenolic acid; mycophenolate mofetil; immunosuppressive monoclonal antibodies, e.g. monoclonal antibodies to leukocyte receptors, e.g. MHC, CD2, CD3, CD4, CD7, DC25, CD28, CTLA4, B7, CD45 or CD58 or your ligands; or other immunomodulating compounds. For anti-inflammatory applications, the compounds of formula I can be used together with anti-inflammatory agents, e.g. corticosteroids. For anti-infective applications, the compounds of formula I can be used in combination with other anti-infective agents, e.g. antiviral drugs or antibiotics. The compounds of formula I are administered by any conventional method, especially enterally, e.g. orally, for example in solutions for drinking, tablets or capsules or parenterally, for example in the form of injectable solutions or suspensions. Suitable unit dosage forms for oral administration include e.g. from 1 to 50 mg of a compound of formula I, usually 1 to 10 mg. Pharmaceutical preparations including the compounds of formula I can be prepared in a conventional manner, e.g. analogous pharmaceutical preparations including rapamycin, e.g. as described in EP A 0 041 795.

Preferably, the pharmaceutical preparations include a compound of formula I and a carrier medium, which medium includes a hydrophilic phase, a lipophilic phase and a surface-active agent. They can be in the form of an emulsion or a microemulsion pre-concentrate. Such emulsions or microemulsion pre-concentrates are described e.g. in GB Patent Publication 2 278 780 A. Preferably the lipophilic phase comprises 10 to 85% by weight of the carrier medium; the surfactant comprises 5 to 80% by weight of the carrier; the hydrophilic phase comprises 10 5 to 50% by weight of the carrier medium. The compound of formula I is preferably present in an amount of 2 to 15% by weight.

A particularly preferred pharmaceutical preparation includes a microemulsion preconcentrated carrier medium comprising

i) a reaction product of a castor oil and ethylene oxide,

ii) a transesterification product of a vegetable oil and glycerol comprising mainly linoleic acid or oleic acid, mono-, di- and triglycerides or a

polyoxyalkylated vegetable oil,

iii) 1,2-propylene glycol, and

iv) ethanol.

According to the foregoing, the present invention also provides:

A. A pharmaceutical preparation comprising a compound of formula I together with a

pharmaceutically acceptable diluent or carrier therefor.

B. A kit or package for use in immunosuppression, inflammation or infections, comprising a pharmaceutical preparation comprising a compound of formula I and a pharmaceutical preparation comprising an immunosuppressive agent or immunomodulatory agent or an anti-inflammatory agent or an anti-infective asthma agent.

C. Use of a compound of formula I for the manufacture of a medicament for preventing or treating acute or chronic organ or tissue transplant rejection, transplant vaculopathies, restenosis, autoimmune diseases, inflammatory disorders or asthma.

It has also surprisingly been found that the compound of formula I where X is OH, i.e. 32(S)-dihydro compounds have an improved activity, e.g. in the above mentioned analyses, and are more stable than the corresponding enantiomers, i.e. 32(R )-dihydro compounds, e.g. when subjected to the following test: The compounds to be tested are incubated in rat serum and their binding affinity for FKBP12 is measured in the MB assay after different incubation times. As the affinity decreases, the nominal ICSO increases. A decrease in affinity is generally attributed to instability of the compound in rat serum.

Claims (10)

1. Connection, characterized by formal in which R 1 is C 1-10 alkyl, C 3-10 alkynyl, hydroxy-C 3-10 alkynyl, R 2 is a residue of formula II; in which R 3 is selected from H, C 1-6 alkyl, hydroxy-C 2-6 alkyl, hydroxy-C 1-6 alkoxy-C 2-6 alkyl or C 1-6 -Alkoxy-C 2-6 -Alkyl; R 4 is H or methyl; Y is O, X is OH or H, R 1 is different from C 1-10 alkyl when X is OH. 2. Compound according to claim 1, characterized in that it has formula Ia in which R 1 is C 3 -ioalk-2-ynyl, R2 is a residue of formula II as defined in claim 1 and Y is O. 3. Compound according to claim 1, characterized in that it is of formula Ib in which R 1 is C 3 -10 alk-2-ynyl, R2 is a residue of formula II as defined in claim 1 and Y is O. 4. Compound according to claim 1, characterized in that it consists of 16-pent-2-ynyloxy-32(S)-dihydro-rapamycin or 16-0-pent-2-ynyl-32(S)-dihydro-40-O-( 2-Hydroxyethyl)rapamycin. 5. Compound according to claim 1, characterized in that it is 32-deoxo-rapamycin or 16-0-pent-2-ynyl-32-deoxo-rapamycin. 6. Process for the preparation of a compound of formula I according to claim 1, characterized in that it comprises a) for the preparation of a compound of formula I where X is H, reductive elimination of carbonyl in position 32 of a compound of formula IVa wherein R[, R.2 and Y are as defined in claim 1, in protected or unprotected form, and, if necessary, removal of protecting groups present; or b) for the preparation of a compound of formula I wherein X is OH, stereoselective reduction of the carbonyl at position 32 of a compound of formula IVa as defined above; or c) converting a compound of formula I wherein R] is alkyl to provide a compound of formula I wherein R 1 is different from alkyl. 7. A compound according to any one of claims 1 to 5, for use as a pharmaceutical agent. 8. Pharmaceutical preparation, characterized in that it comprises a compound according to any one of claims 1 to 5, together with a pharmaceutically acceptable diluent or a carrier for this. 9. Kit or packaging for use in immunosuppression, inflammation or infections, characterized in that it comprises a pharmaceutical preparation comprising a compound according to any one of claims 1 to 5 and a pharmaceutical preparation comprising an immunosuppressive agent or immunomodulating drug or an anti-inflammatory drug or an anti-infective asthma drug. 10. Use of a compound according to any one of claims 1 to 5 for the manufacture of a medicament for preventing or treating acute or chronic organ or tissue transplant rejection, transplant vasculopathies, restenosis, autoimmune diseases, inflammatory disorders or asthma.